Mice
returned
to
holding
cages,
arena
cleaned
and
system
readied
for
hole
board
test

-

-

-

-

4

Mice
placed
in
the
center
arena
of
an
exploratory
hole
board
apparatus
to
measure
activity
exploratory
habits,
and
anxiety-like
behaviors

hole
board
test

hole
board
arena,
activity
tracker

10
min

rearing
and
nose
poke
activities

5

Mice
returned
to
home
cages
and
arena
platform
cleaned
and
system
re
died
for
the
next
mouse

-

-

-

-

Equipment
and
supplies

Open
field
arena
made
of
a
clear
acrylic
(40
X
40
X
30
cm)
placed
inside
a
frame
containing
evenly
spaced
photocells
and
receptors
making
a
grid
of
infrared
photobeams
from
the
front
to
the
back
and
from
the
left
to
the
right
(AccuScan
Instruments
Inc.,
Columbus,
Ohio)

Figure
2.
Schematic
layout
of
the
open
field
chamber
without
and
with
the
hole
board
setup.

Reagents
and
solutions

Food/reward
pellets

Disinfectant
and
cleaning
solutions

Acclimation
to
test
conditions

Testing
is
conducted
during
the
light
phase
between
08:00
and
16:00
h.
Mice
are
transported
from
the
vivarium
into
the
test
room
and
are
allowed
to
habituate
for
at
least
30
min
before
testing.

Procedures

I.
Open
field
testActivity
assessment
is
conducted
in
4
square
arenas
constructed
of
clear
acrylic
panels
(see
Figs.
1
and
2).
Mouse
activity
is
measured
via
a
grid
of
invisible
infrared
light
beams.
A
number
of
equally
spaced
beams
traverse
the
mouse
cage
front
to
back
and
an
equal
number
of
beams
traverse
the
same
cage
from
left
to
right.
Any
movement
made
by
a
mouse
within
the
VersaMax
monitor
break
through
these
light
beams.
Thus
revealing
the
mouse’s
position
in
the
(X-Y)
plane.
Vertical
sensors
monitor
rearing
and
jumping
activity.
The
Versamax
software
analyzer
collects
the
beam
status
information
from
the
activity
monitor
and
subjects
it
to
rapid
analysis.
Each
time
the
analyzer
receives
the
beam
status,
it
is
able
to
determine
the
position
of
the
mouse
at
50
times
per
second.
At
this
speed
stereotypic
activity,
as
well
as
hyperactivity,
where
a
mouse
transverses
the
entire
cage
in
fractions
of
a
second
is
readily
monitored.
a.
Each
of
the
4
arenas
is
mounted
within
specially
designed
sound
attenuating
shells
constructed
of
polypropylene
and
regular
and
expanded
PVC.
Besides
keeping
outside
noise
to
a
minimum,
these
chambers
also
keep
out
external
light
and
odors.
b.
Before
testing
the
VersaMax
monitoring
system
is
automatically
checked
and
configured
according
to
manufacturer's
protocol
and
is
illuminated
with
a
red
7.5W
light
bulb.c.
With
the
automatic
tracking
system
turned
ON,
each
mouse
is
placed
in
the
center
of
the
arena
(Fig.
3)
and
allowed
to
move
freely
about
the
novel
environment
for
10
min
(600
s).
The
VersaMax
Analyzer
collects
the
beam
status
information
50
times
per
seconds,
a
speed
sufficient
to
reveal
stereotypic
activity
and
hyperactivity.e.
Horizontal,
vertical,
stereotypical,
total
distance
traveled,
thigmotaxis,
and
center
activity
measurements
are
extracted
from
automatically
recorded
data.d.
At
the
conclusion
of
each
trial
the
mouse
is
temporarily
removed
from
the
arena,
returned
to
its
holding
cage,
and
the
system
readied
for
subsequent
hole
board
test.f.
In
between
mice,
the
surface
of
the
arena
is
disinfected
with
70%
ethanol.

Figure
3.
Mice
are
placed
in
the
center
of
the
arena.

II.
Hole
board
testThe
open
field
system
is
adjusted
to
study
hole
poke/nose
poke
behavior
using
a
baited
or
non-baited
hole
board
placed
in
the
standard
mouse
cage.
Like
the
open
field,
the
exploratory
hole-board
test
is
used
to
determine
general
activity
levels
and
locomotor
activity,
as
well
as
exploratory
habits,
and
anxiety-like
behavior
in
a
novel
environment.
a.
An
elevated
platform
with
16
equally
spaced
holes
is
placed
within
the
open
field
system
according
manufacturer's
instruction
(see
Fig.
2).
Each
hole
is
large
enough
for
a
mouse
to
puts
its
head
fully
into
the
hole
without
being
able
to
pass
completely
through
to
the
other
side.
b.
A
tray
of
small
food/reward
cups
with
a
removable
screen
is
placed
under
each
hole.
Typically
a
small
food/reward
pellet
is
placed
in
each
of
the
16
cups.
Only
when
a
screen
is
placed
over
the
cup,
that
the
mouse
is
able
to
see
and
smell
the
food/reward,
but
cannot
retrieve
the
reward.
c.
A
designated
cup
is
chosen
as
the
target
hole
and
then
left
uncovered,
while
all
other
cups
are
covered
with
screens.
d.
Light
beam
sensors
are
then
positioned
between
the
platform
and
the
food
cups.
These
sensors
register
the
specific
hole
a
mouse
is
investigating.
e.
A
mouse
is
placed
on
the
platform
and
allowed
to
investigate
holes
at
will
for
10
min
(600
s).
f.
The
duration
and
location
of
the
query
for
each
hole
investigated
is
automatically
recorded,
and
only
visits
to
the
target
hole
are
rewarded.g.
Rearing
and
nose
poke
activity
measurements
are
extracted
from
automatically
recorded
data.h.
In
between
mice,
the
surface
of
the
arena
is
disinfected
with
70%
ethanol.

Definitions
&
data
generated
variables

HACTV:
The
total
number
of
beam
interruptions
that
occurred
in
the
horizontal
sensor
during
a
given
sample
period.

TOTDIST:
Total
distance
traveled
is
not
the
same
as
"horizontal
activity"
counts.
If
the
animal
travels
from
one
corner
to
the
diagonally
opposite
corner,
he
will
generate
the
same
"horizontal
activity"
count
regardless
of
path
traveled.
The
distance
traveled
however,
is
dependent
on
the
path
that
the
animal
takes.
Hence,
"total
distance"
is
a
more
accurate
indicator
of
ambulatory
activity!
The
number
indicates
in
centimeters
the
distance
traveled
by
the
animal
in
a
given
sample
period.

MOVNO:
Number
of
discrete
horizontal
movements.
Each
time
a
break
in
ambulatory
activity
occurs
for
a
period
greater
than
1
s,
this
variable
is
incremented
by
1.
This
indicates
the
number
of
separate
horizontal
movements
executed
by
the
animal
in
a
given
sample
period.
Individual
movements
are
separated
from
each
other
by
a
rest
period
of
at
least
1
s.

MOVTIME:
As
long
as
the
animal
is
ambulating,
this
variable
is
incremented.
If
the
animal
is
non-ambulatory
for
more
than
1
s,
this
variable
is
no
longer
incremented.
Thus
it
corresponds
to
the
amount
of
time
the
animal
was
in
ambulation
during
a
given
sample
period.
Does
not
include
stereotypy
time.

RESTIME:
It
is
computed
as
the
difference
between
sample
time
and
time
spent
moving.

VACTV:
The
total
number
of
beam
interruptions
that
occurred
in
the
vertical
sensor
during
a
given
sample
period.

VMOVNO:
Each
time
the
animal
rears
up,
this
variable
is
incremented
by
1.
The
animal
must
go
below
the
level
of
the
vertical
sensor
for
at
least
1
s
before
the
next
rearing
can
be
registered.

VTIME:
When
the
animal
activates
the
vertical
sensor
by
rearing,
this
variable
starts
incrementing
and
continues
to
increment
until
the
animal
goes
below
the
level
of
the
vertical
sensor.

STRCNT:
If
the
animal
breaks
the
same
beam
(or
set
of
beams)
repeatedly
then
the
monitor
considers
that
the
animal
is
exhibiting
stereotypy.
This
typically
happens
during
grooming,
head
bobbing,
etc.
Stereotypy
count
is
the
number
of
beam
breaks
that
occur
during
this
period
of
stereotypic
activity.

STRNO:
This
corresponds
to
the
number
of
times
the
monitor
observed
stereotypic
behavior
in
the
animal.
A
break
in
stereotypy
of
1
s
or
more
is
required
to
separate
one
stereotypic
episode
from
the
next.

STRTIME:
The
total
amount
of
time
that
stereotypic
behavior
is
exhibited.

MRGDIST:
The
distance
(in
centimeters)
an
animal
travels
while
in
the
margin
or
corners
of
the
cage.

MRGTIME:
Time
spent
by
the
animal
in
close
proximity
(within
1
cm)
to
the
walls
of
the
cage

CTRDIST:
The
distance
(in
centimeters)
an
animal
travels
while
in
the
center
of
the
cage.